The study of transcriptional regulation of necrotrophic effector genes ToxA and Tox3 in the wheat pathogen Parastagonospora nodorum
Date
2017
Authors
Lin, Shao-Yu
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Abstract
Parastagonospora nodorum (P. nodorum) is an important
necrotrophic fungus on wheat causing significant yield losses in
Australia per annum. It is now widely recognized that P. nodorum
causes disease by secreting necrotrophic effectors (NEs) which
induce necrosis on susceptible wheat leaves through an inverse
gene for gene manner. Two of the NEs described are ToxA and Tox3.
Previous reports have shown that ToxA and Tox3 are highly
upregulated during infection. However, how these effectors are
regulated and expressed is largely unclear. To better understand
the transcriptional regulation of ToxA and Tox3, I conducted
functional analyses on their promoter regions to identify the key
factors for the regulation in P. nodorum.
Being a necrotroph, P. nodorum tends to acquire nutrients from
surrounding environment. It is therefore hypothesized that P.
nodorum senses nutrients during infection to trigger the
expression of the NE genes. By analyzing the ability of promoters
to trigger gene expression under different nutrient condition, it
was found that nitrogen sources such as nitrate and ammonium
involved in expression of Tox3. On the other hand, the expression
of ToxA was extremely weak in all tested media. Moreover, through
promoter deletion analysis, I was unable to identify a
cis-regulatory element other than TATA box on the ToxA promoter.
DNase I footprinting and yeast one-hybrid assay also did not
identify protein candidates interacting with the ToxA promoter.
Interestingly, the ToxA promoter showed ability to induce strong
expression in the tested media after replacing the ToxA gene with
GFP. In contrast, much lower GFP transcripts were observed in the
presence of ToxA CDS when expressed under the same ToxA promoter.
These results imply that ToxA may be regulated at transcriptional
elongation or post-transcriptional stage.
In previous studies, it is recognized that Tox3 was regulated by
transcription factors. Nevertheless, none of the studies showed
evidence of direct interaction. In this study, the promoter and
expression of Tox3 was characterized. A combination of promoter
deletion and DNase I footprinting experiments identified a 25 bp
region (Tox3-URE1) on the Tox3 promoter as being required for
transcription. Subsequent yeast one-hybrid analysis identified a
C2H2 zinc finger transcription factor PnCon7 specifically
interacting with Tox3-URE1. Silencing of PnCon7 resulted in the
down-regulation of Tox3 in a dose dependent manner. Moreover,
pathogenicity and expressions of two other necrotrophic effectors
(ToxA and Tox1) were identified to be affected in an indirect
way.
Collectively, in my PhD study, I did not identify cis-regulatory
elements or transcription factors responsible for the expression
of ToxA. Alternatively, ToxA was found to be regulated at
transcriptional elongation or post-transcriptional stage. On the
other hand, the expression of Tox3 was largely upregulated when
encountering specific nitrogen source. Furthermore, a C2H2 zinc
finger transcription factor PnCon7 was found to specifically bind
to the Tox3 promoter and regulated its expression through a dose
dependent manner. This study highlights the latest understanding
of how NEs are regulated at the transcriptional level in fungi
and provides a fundamental research for future studies in
transcriptional regulation of fungal virulence genes.
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Parastagonospora nodorum, necrotrophic effector, ToxA, Tox3, wheat, transcription factor, PnCon7, PnPf2, transcriptional regulation, alternative splicing, pathogen, infection, plant, pathogenicity, fungus
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